TW202421669A - Antibodies specifically binding to asm protein - Google Patents
Antibodies specifically binding to asm protein Download PDFInfo
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- TW202421669A TW202421669A TW112145060A TW112145060A TW202421669A TW 202421669 A TW202421669 A TW 202421669A TW 112145060 A TW112145060 A TW 112145060A TW 112145060 A TW112145060 A TW 112145060A TW 202421669 A TW202421669 A TW 202421669A
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Abstract
本發明涉及一種與酸性神經鞘磷脂酶（acid sphingomyelinase，ASM）蛋白特異性結合的抗體及其用途。具體地，本發明的抗體或其抗原結合片段以特異性地高的結合力與酸性神經鞘磷脂酶蛋白結合，從而可以用於檢測酸性神經鞘磷脂酶蛋白或診斷由酸性神經鞘磷脂酶蛋白的過表現引起的疾病。The present invention relates to an antibody that specifically binds to an acid sphingomyelinase (ASM) protein and its use. Specifically, the antibody or its antigen-binding fragment of the present invention binds to the acid sphingomyelinase protein with a specific high binding force, and can be used to detect the acid sphingomyelinase protein or diagnose diseases caused by the overexpression of the acid sphingomyelinase protein.
Description
本發明涉及一種與酸性神經鞘磷脂酶（acid sphingomyelinase，ASM）蛋白特異性結合的抗體。The present invention relates to an antibody which specifically binds to acid sphingomyelinase (ASM) protein.
鞘脂代謝調節正常的細胞信號轉導，鞘脂代謝的異常變化對包括阿茲海默病在內的各種神經退化性疾病產生影響。酸性神經鞘磷脂酶（acid sphingomyelinase，ASM）蛋白作爲調節鞘脂代謝的酶，是一種在幾乎所有類型的細胞中表現的蛋白質，在鞘脂代謝以及細胞膜周轉（turnover）中發揮重要作用。Sphingolipid metabolism regulates normal cell signal transduction, and abnormal changes in sphingolipid metabolism affect various neurodegenerative diseases including Alzheimer's disease. Acid sphingomyelinase (ASM) protein, as an enzyme regulating sphingolipid metabolism, is a protein expressed in almost all types of cells and plays an important role in sphingolipid metabolism and cell membrane turnover.
與正常人相比，酸性神經鞘磷脂酶蛋白的活性在如阿茲海默病等神經退化性疾病患者的大腦中顯著增加。對此，韓國專利授權第10-1521117號中公開了當抑制過表現的酸性神經鞘磷脂酶蛋白的活性或抑制酸性神經鞘磷脂酶蛋白的表現時，可以抑制β澱粉樣蛋白的積累並改善學習能力以及記憶力，從而治療神經退化性疾病。並且，最近已知酸性神經鞘磷脂酶蛋白的活性在如抑鬱症等神經疾病中也增加，因此抑制上述酸性神經鞘磷脂酶蛋白的表現或活性具有改善抑鬱症的效果。Compared with normal people, the activity of acidic neurosphingomyelinase protein is significantly increased in the brains of patients with neurodegenerative diseases such as Alzheimer's disease. In this regard, Korean Patent No. 10-1521117 discloses that when the activity of overexpressed acidic neurosphingomyelinase protein is inhibited or the expression of acidic neurosphingomyelinase protein is inhibited, the accumulation of β-amyloid protein can be inhibited and learning ability and memory can be improved, thereby treating neurodegenerative diseases. In addition, it has recently been known that the activity of acidic neurosphingomyelinase protein is also increased in neurological diseases such as depression, so inhibiting the expression or activity of the above-mentioned acidic neurosphingomyelinase protein has the effect of improving depression.
然而，還沒有開發出直接抑制酸性神經鞘磷脂酶蛋白的表現或活性的物質，但是，已鑑定幾種間接抑制酸性神經鞘磷脂酶蛋白的表現的抑制劑。例如，有用於治療抑鬱症的三環類抗抑鬱藥（tricyclic antidepressant），其包括阿米替林（amitriptyline）、地昔帕明（desipramine）、丙咪嗪（mipramine）等。雖然這些三環類抗抑鬱藥並不是開發爲酸性神經鞘磷脂酶蛋白抑制劑，但通過各種研究結果證明表現出酸性神經鞘磷脂酶蛋白抑制效果。三環類抗抑鬱藥的主要藥理機制爲通過抑制神經細胞中神經傳導物質的再攝取來增加其活性，確認其作爲酸性神經鞘磷脂酶抑制劑的作用爲次要作用。然而，由於三環類抗抑鬱作用於神經系統以及神經細胞而可能導致視力模糊、光敏感性增加、嘔吐等副作用。However, no substance that directly inhibits the expression or activity of acidic sphingomyelinase protein has been developed, but several inhibitors that indirectly inhibit the expression of acidic sphingomyelinase protein have been identified. For example, there are tricyclic antidepressants that are useful for treating depression, including amitriptyline, desipramine, and mipramine. Although these tricyclic antidepressants were not developed as acidic sphingomyelinase protein inhibitors, they have been shown to exhibit acidic sphingomyelinase protein inhibitory effects through various research results. The main pharmacological mechanism of tricyclic antidepressants is to increase the activity of neurotransmitters by inhibiting their reuptake in nerve cells, and their role as acidic sphingomyelinase inhibitors is confirmed as a secondary effect. However, since tricyclic antidepressants act on the nervous system and nerve cells, they may cause side effects such as blurred vision, increased photosensitivity, and vomiting.
發明所欲解決之問題Invent the problem you want to solve
本發明的一目的在於提供與酸性鞘磷脂酶蛋白特異性結合的抗體或其抗原結合片段。One object of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to acid sphingomyelinase protein.
本發明的再一目的在於提供上述抗體或其抗原結合片段的製備方法。Another object of the present invention is to provide a method for preparing the above-mentioned antibody or antigen-binding fragment thereof.
本發明的還有一目的在於提供上述抗體或其抗原結合片段的酸性鞘磷脂酶蛋白檢測用途。Another object of the present invention is to provide the use of the above-mentioned antibody or its antigen-binding fragment for detecting acid sphingomyelinase protein.
解決問題之技術手段Technical means to solve the problem
為了實現上述目的，本發明提供與酸性鞘磷脂酶（acid sphingomyelinase）蛋白特異性結合的抗體或其抗原結合片段。In order to achieve the above object, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to an acid sphingomyelinase protein.
並且，本發明提供對上述抗體或其抗原結合片段進行編碼的核酸。Furthermore, the present invention provides a nucleic acid encoding the above-mentioned antibody or an antigen-binding fragment thereof.
並且，本發明提供包含上述核酸的表現載體。Furthermore, the present invention provides an expression vector comprising the above nucleic acid.
並且，本發明提供包含上述核酸或表現載體的宿主細胞。Furthermore, the present invention provides a host cell comprising the above-mentioned nucleic acid or expression vector.
並且，本發明提供酸性鞘磷脂酶蛋白特異性結合的抗體或其抗原結合片段的生產方法，其包含通過培養上述宿主細胞來生產抗體或其抗原結合片段的步驟。Furthermore, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to acid sphingomyelinase protein, which comprises the step of producing the antibody or the antigen-binding fragment thereof by culturing the above-mentioned host cells.
並且，本發明提供包含上述抗體或其抗原結合片段的酸性鞘磷脂酶蛋白檢測用組合物及試劑盒。Furthermore, the present invention provides a composition and a reagent kit for detecting acid sphingomyelinase protein comprising the above-mentioned antibody or an antigen-binding fragment thereof.
進而，本發明提供酸性鞘磷脂酶蛋白檢測方法，其包含使上述抗體或其抗原結合片段與試樣產生反應的步驟。Furthermore, the present invention provides a method for detecting acid sphingomyelinase protein, which comprises the step of reacting the above-mentioned antibody or antigen-binding fragment thereof with a sample.
對照先前技術之功效Comparison with the efficacy of previous technologies
對照先前技術之功效本發明的抗體或其抗原結合片段以特異性地高的結合力與酸性神經鞘磷脂酶蛋白結合，從而可以用於檢測酸性神經鞘磷脂酶蛋白或診斷由酸性神經鞘磷脂酶蛋白的過表現引起的疾病。Compared with the efficacy of the prior art, the antibody or antigen-binding fragment thereof of the present invention binds to acidic sphingomyelinase protein with high specificity, and thus can be used to detect acidic sphingomyelinase protein or diagnose diseases caused by overexpression of acidic sphingomyelinase protein.
以下，詳細說明本發明。The present invention is described in detail below.
本發明提供與酸性鞘磷脂酶（ASM，acid sphingomyelinase）蛋白特異性結合的抗體或其抗原結合片段。The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to an acid sphingomyelinase (ASM) protein.
在本說明書中使用的術語“酸性鞘磷脂酶（ASM，acid sphingomyelinase）蛋白”意味著作為調節鞘脂代謝的SMase（sphingomyelinase）族之一的酶。上述酸性鞘磷脂酶蛋白促進將鞘磷脂（sphingomyelin）分解成神經醯胺（ceramide）以及磷酸膽鹼（phosphorylcholine）的過程，可根據表示最佳酶活性的pH來分為鹼性、中性或酸性。The term "acid sphingomyelinase (ASM) protein" used in this specification means an enzyme that is one of the SMase (sphingomyelinase) family that regulates sphingolipid metabolism. The acid sphingomyelinase protein promotes the process of breaking down sphingomyelin into ceramide and phosphorylcholine, and can be classified into alkaline, neutral or acidic according to the pH that shows the best enzyme activity.
上述酸性神經鞘磷脂酶蛋白可以包括常規技術領域公知的所有類型的酸性神經鞘磷脂酶蛋白。具體地，上述酸性神經鞘磷脂酶蛋白可以來源於哺乳動物，更具體地，可以來源於人類、猴、大鼠或小鼠。並且，上述酸性神經鞘磷脂酶蛋白可以包括常規技術領域已知爲酸性神經鞘磷脂酶蛋白的所有胺基酸序列。例如，上述酸性神經鞘磷脂酶蛋白可以爲由如序列139所示的胺基酸序列組成的多肽或編碼其的核酸。The above-mentioned acidic neurosphingomyelinase protein can include all types of acidic neurosphingomyelinase proteins known in the conventional art. Specifically, the above-mentioned acidic neurosphingomyelinase protein can be derived from mammals, more specifically, can be derived from humans, monkeys, rats or mice. And, the above-mentioned acidic neurosphingomyelinase protein can include all amino acid sequences known as acidic neurosphingomyelinase proteins in the conventional art. For example, the above-mentioned acidic neurosphingomyelinase protein can be a polypeptide composed of an amino acid sequence as shown in sequence 139 or a nucleic acid encoding it.
上述酸性神經鞘磷脂酶蛋白可以在如序列139所示的胺基酸序列中被添加、删除或取代一個或其以上的胺基酸，是要其保持相同或與此相應的生物活性即可。在此情况下，胺基酸的取代可以爲在不影響或輕微影響整個蛋白質的電荷，即極性或疏水性的範圍內進行的保守取代。並且，上述酸性神經鞘磷脂酶蛋白可以與如序列139所示的胺基酸序列具有80%以上、90%以上、95%以上、97%以上或99%以上的同源性。The acidic sphingomyelinase protein may be added, deleted or substituted with one or more amino acids in the amino acid sequence shown in SEQ ID NO: 139, as long as the same or corresponding biological activity is maintained. In this case, the amino acid substitution may be a conservative substitution that does not affect or slightly affects the charge of the entire protein, i.e., polarity or hydrophobicity. Furthermore, the acidic sphingomyelinase protein may have a homology of more than 80%, more than 90%, more than 95%, more than 97% or more than 99% with the amino acid sequence shown in SEQ ID NO: 139.
在本說明書中使用的術語“抗體（antibody）”意味著通過與抗原相結合來妨礙抗原的作用或去除抗原的免疫蛋白。上述抗體可包括普通技術領域中所包含的所有種類的抗體。具體地，上述抗體可包括IgM、IgD、IgG、IgA以及IgE，分別包括由作為對重鏈不變區域進行編碼的基因的μ、δ、γ、α以及ε組成的重鏈。通常，在抗體技術中主要使用IgG，這重新由IgG1、IgG2、IgG3或IgG4的同型（isotype）組成，它們各自的結構以及功能特性可不同。上述抗體可包括將源自非-人抗體的最小序列包含在其中的人化抗體、由源自人的序列組成的人抗體或混合源自不同物種的序列的嵌合抗體等的全部。The term "antibody" used in this specification means an immune protein that interferes with the action of an antigen or removes an antigen by binding to an antigen. The above-mentioned antibodies may include all types of antibodies included in the ordinary technical field. Specifically, the above-mentioned antibodies may include IgM, IgD, IgG, IgA and IgE, respectively including a heavy chain composed of μ, δ, γ, α and ε as genes encoding invariant regions of the heavy chain. Generally, IgG is mainly used in antibody technology, which is composed of isotypes of IgG1, IgG2, IgG3 or IgG4, and their respective structures and functional properties may be different. The above-mentioned antibodies may include all of humanized antibodies containing minimal sequences derived from non-human antibodies, human antibodies composed of sequences derived from humans, or chimeric antibodies that mix sequences derived from different species.
上述IgG可以形成由2個約50kDa的重鏈（heavy chain）蛋白以及2個約25kDa的輕鏈（light chain）蛋白形成Y字形的非常穩定的結構（約150kDa）。組成抗體的輕鏈以及重鏈可以分爲抗體之間具有不同胺基酸序列的可變區（variable region）以及具有相同胺基酸序列的恒定區（constant region）。在此情况下，重鏈恒定區包含CH1、鉸鏈（hinge，H）、CH2以及CH3結構域，每個結構域由2個β折疊（β-sheet）組成，可以通過分子內二硫鍵（disulfide bond）連接。在此情况下，重鏈以及輕鏈的2個可變區結合形成抗原結合位點（antigen binding site），上述抗原結合位點可以存在於2個Y字形臂中的各一個。全長的抗體中能夠與抗原結合的部分被稱爲抗原結合片段（antibody binding fragment，Fab），不能與抗原結合的部分被稱爲可結晶片段（crystallizable fragment，Fc），Fab以及Fc可以通過鉸鏈連接。The above IgG can form a very stable structure (about 150kDa) in the shape of a Y, which is composed of two heavy chain proteins of about 50kDa and two light chain proteins of about 25kDa. The light chain and heavy chain that constitute the antibody can be divided into a variable region with different amino acid sequences between antibodies and a constant region with the same amino acid sequence. In this case, the heavy chain constant region includes CH1, hinge (H), CH2 and CH3 domains, each of which is composed of two β-sheets and can be connected by intramolecular disulfide bonds. In this case, the two variable regions of the heavy chain and light chain combine to form an antigen binding site, and the above antigen binding site can exist in one of the two Y-shaped arms. The part of the full-length antibody that can bind to the antigen is called the antigen-binding fragment (Fab), and the part that cannot bind to the antigen is called the crystallizable fragment (Fc). Fab and Fc can be connected by hinges.
在本發明一實施例中，上述IgG可以爲人源IgG，具體地，可以包含由如序列137所示的胺基酸序列組成的人IgG重鏈恒定區以及由如序列138所示的胺基酸序列組成的人輕鏈λ恒定區。並且，上述人源IgG可以包含由如序列140所示的鹼基序列組成的人IgG重鏈恒定區以及由如序列141所示的鹼基序列組成的人輕鏈λ恒定區。In one embodiment of the present invention, the above IgG may be human IgG, specifically, it may include a human IgG heavy chain constant region composed of an amino acid sequence as shown in SEQ ID NO: 137 and a human light chain λ constant region composed of an amino acid sequence as shown in SEQ ID NO: 138. Furthermore, the above human IgG may include a human IgG heavy chain constant region composed of a base sequence as shown in SEQ ID NO: 140 and a human light chain λ constant region composed of a base sequence as shown in SEQ ID NO: 141.
本發明的抗體不僅包括全長的抗體，還包括其的抗原結合片段。具體地，上述抗原結合片段可意味著除了起到向細胞或補體傳遞與抗原之間的結合刺激的功能的Fc的部分。作為一例，抗體的抗原結合片段可包括Fab、scFv、F（ab）2以及Fv等的全部，還可包括單域抗體（single domain antibody）或微型抗體（minibody）等的第三代抗體切片。The antibodies of the present invention include not only full-length antibodies but also antigen-binding fragments thereof. Specifically, the antigen-binding fragments may mean the part of Fc that has the function of transmitting the binding stimulation between the antigen and the cell or complement. As an example, the antigen-binding fragments of the antibody may include all of Fab, scFv, F(ab)2 and Fv, and may also include third-generation antibody fragments such as single domain antibodies or minibodies.
例如，上述抗體或其抗原結合片段可以包含：重鏈可變區，組成自：由如序列89所示的胺基酸序列組成的重鏈CDR1、由如序列90所示的胺基酸序列組成的多肽中的5個以下胺基酸被取代的重鏈CDR2以及由如序列91所示的胺基酸序列組成的重鏈CDR3；以及輕鏈可變區，組成自：由如序列92所示的胺基酸序列組成的輕鏈CDR1、由如序列93所示的胺基酸序列組成的多肽或上述多肽中的5個以下胺基酸被取代的輕鏈CDR2以及由序列94所示的胺基酸序列組成的多肽或上述多肽中的2個以下胺基酸被取代的輕鏈CDR3。For example, the above-mentioned antibody or its antigen-binding fragment may comprise: a heavy chain variable region, composed of: a heavy chain CDR1 composed of the amino acid sequence shown in sequence 89, a heavy chain CDR2 in which no more than 5 amino acids in a polypeptide composed of the amino acid sequence shown in sequence 90 are substituted, and a heavy chain CDR3 composed of the amino acid sequence shown in sequence 91; and a light chain variable region, composed of: a light chain CDR1 composed of the amino acid sequence shown in sequence 92, a light chain CDR2 in which no more than 5 amino acids in a polypeptide composed of the amino acid sequence shown in sequence 93 or the above polypeptide are substituted, and a light chain CDR3 in which no more than 2 amino acids in a polypeptide composed of the amino acid sequence shown in sequence 94 or the above polypeptide are substituted.
上述術語“CDR（complementarity determining region）”為作為每個抗體在抗體的重鏈以及輕鏈可變區域內具有不同胺基酸序列的部位的高變區（hypervariable region），意味著實際與抗原相結合的部位。在抗體的立體結構中，CDR以環狀（loop）位於抗體的表面，可在環下方存在結構性地對其進行支撐的FR（framework region）。重鏈以及輕鏈均分別具有三個環結構，這些六個環結構可相合併與抗原直接接觸。方便起見，可將作為具有上述六個環結構的抗原結合部位的CDR分別稱作重鏈CDR1、重鏈CDR2、重鏈CDR3、輕鏈CDR1、輕鏈CDR2或輕鏈CDR3。The term "CDR (complementarity determining region)" is a hypervariable region that has different amino acid sequences in the heavy chain and light chain variable regions of each antibody, and means the site that actually binds to the antigen. In the three-dimensional structure of the antibody, CDR is located on the surface of the antibody in a loop, and there may be a FR (framework region) under the loop to structurally support it. The heavy chain and light chain each have three loop structures, and these six loop structures can combine and directly contact the antigen. For convenience, the CDRs that are the antigen binding sites having the above six loop structures may be referred to as heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2 or light chain CDR3.
在本發明的抗體或其抗原結合片段中，在由如序列90所示的胺基酸序列組成的多肽中的5個以下胺基酸被取代的重鏈CDR2中，可以從上述多肽的N-末端起選自由第2位、第4位至第10位、第12位、第13位以及第17位胺基酸組成的組中的5個以下胺基酸被取代。In the antibody or antigen-binding fragment thereof of the present invention, in the heavy chain CDR2 in which no more than 5 amino acids in a polypeptide consisting of an amino acid sequence as shown in SEQ ID NO: 90 are substituted, no more than 5 amino acids selected from the group consisting of the 2nd, 4th to 10th, 12th, 13th and 17th amino acids from the N-terminus of the above-mentioned polypeptide can be substituted.
例如，從上述由如序列90所示的胺基酸序列組成的多肽的N-末端起第2位、第4位、第6位、第7位、第9位、第12位或第13位胺基酸可以被中性胺基酸取代，具體地，上述中性胺基酸可以包括丙胺酸（alanine）、甘胺酸（glycine）、亮胺酸（leucine）、異亮胺酸（isoleucine）、脯胺酸（proline）、纈胺酸（valine）、***酸（phenylalanine）、色胺酸（tryptophan）、酪胺酸（tyrosine）、絲胺酸（serine）、蘇胺酸（threonine）、半胱胺酸（cysteine）、蛋胺酸（methionine）、天冬醯胺（asparagine）以及穀胺醯胺（glutamine）。更具體地，作爲從上述N-末端起第2位胺基酸的異亮胺酸可以被亮胺酸、纈胺酸或蘇胺酸取代，作爲第4位胺基酸的酪胺酸可以被***酸或色胺酸取代，作爲第6位胺基酸的絲胺酸可以被甘胺酸或天冬醯胺取代，作爲第7位胺基酸的甘胺酸可以被纈胺酸取代，作爲第9位胺基酸的異亮胺酸可以被脯胺酸、丙胺酸或纈胺酸取代，作爲第12位胺基酸的丙胺酸可以被絲胺酸取代，作爲第13位胺基酸的天冬胺酸可以被天冬醯胺或丙胺酸取代。另一方面，從上述由如序列90所示的胺基酸序列組成的多肽的N-末端起第10位胺基酸可以被鹼性胺基酸取代，具體地，上述鹼性胺基酸可以包括精胺酸（arginine）、組胺酸（histidine）以及賴胺酸（lysine）。更具體地，作爲從上述N-末端起第10位胺基酸的酪胺酸可以被精胺酸取代。並且，從上述由如序列90所示的胺基酸序列組成的多肽的N-末端起第17位胺基酸可以被酸性胺基酸取代，具體地，上述酸性胺基酸可以包括天冬胺酸（aspartic acid）以及谷胺酸（glutamic acid）。更具體地，作爲從上述N-末端起第17位胺基酸的甘胺酸可以被天冬胺酸或谷胺酸取代。並且，從上述由如序列90所示的胺基酸序列組成的多肽的N-末端起第5位胺基酸可以爲中性或酸性胺基酸，上述中性或酸性胺基酸可以如上所述。更具體地，作爲從上述N-末端起第5位胺基酸的甘胺酸可以被丙胺酸、天冬胺酸、絲胺酸或脯胺酸取代。另外，從上述由如序列90所示的胺基酸序列組成的多肽的N-末端起第8位胺基酸可以爲中性、酸性或鹼性胺基酸，上述中性、酸性或鹼性胺基酸可以如上所述。更具體地，作爲從上述N-末端起第8位胺基酸的天冬醯胺可以被天冬胺酸、賴胺酸、異亮胺酸、蘇胺酸、纈胺酸、甘胺酸或酪胺酸取代。For example, the 2nd, 4th, 6th, 7th, 9th, 12th or 13th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence as shown in SEQ ID NO:90 may be substituted with a neutral amino acid. Specifically, the neutral amino acid may include alanine, glycine, leucine, isoleucine, proline, valine, phenylalanine, tryptophan, tyrosine, serine, threonine, cysteine, methionine, asparagine and glutamine. More specifically, isoleucine as the second amino acid from the N-terminus may be substituted with leucine, valine or threonine, tyrosine as the fourth amino acid may be substituted with phenylalanine or tryptophan, serine as the sixth amino acid may be substituted with glycine or asparagine, glycine as the seventh amino acid may be substituted with valine, isoleucine as the ninth amino acid may be substituted with proline, alanine or valine, alanine as the twelfth amino acid may be substituted with serine, and aspartic acid as the thirteenth amino acid may be substituted with asparagine or alanine. On the other hand, the 10th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 90 may be substituted with a basic amino acid, and specifically, the basic amino acid may include arginine, histidine, and lysine. More specifically, tyrosine, which is the 10th amino acid from the N-terminus, may be substituted with arginine. Furthermore, the 17th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 90 may be substituted with an acidic amino acid, and specifically, the acidic amino acid may include aspartic acid and glutamic acid. More specifically, glycine, which is the 17th amino acid from the N-terminus, may be substituted with aspartic acid or glutamic acid. Furthermore, the 5th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence as shown in Sequence 90 can be a neutral or acidic amino acid, and the neutral or acidic amino acid can be as described above. More specifically, the glycine as the 5th amino acid from the N-terminus can be replaced by alanine, aspartic acid, serine or proline. In addition, the 8th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence as shown in Sequence 90 can be a neutral, acidic or alkaline amino acid, and the neutral, acidic or alkaline amino acid can be as described above. More specifically, asparagine as the 8th amino acid from the N-terminus can be replaced by aspartic acid, lysine, isoleucine, threonine, valine, glycine or tyrosine.
在本發明一實施例中，上述由如序列90所示的胺基酸序列組成的多肽中的5個以下胺基酸被取代的重鏈CDR2可以爲由分別如序列95至序列111所示的胺基酸序列組成的多肽。In one embodiment of the present invention, the heavy chain CDR2 in which less than 5 amino acids in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 90 are substituted may be a polypeptide consisting of the amino acid sequences shown in SEQ ID NO: 95 to SEQ ID NO: 111, respectively.
在本發明的抗體或其抗原結合片段中，在由如序列93所示的胺基酸序列組成的多肽中的5個以下胺基酸被取代的輕鏈CDR2中，可以從上述多肽的N-末端起選自由第3位至第7位胺基酸組成的組中的4個以下胺基酸被取代。In the antibody or antigen-binding fragment thereof of the present invention, in the light chain CDR2 in which no more than 5 amino acids in a polypeptide consisting of an amino acid sequence as shown in SEQ ID NO: 93 are substituted, no more than 4 amino acids selected from the group consisting of amino acids at positions 3 to 7 from the N-terminus of the above-mentioned polypeptide can be substituted.
例如，從上述由如序列93所示的胺基酸序列組成的多肽的N-末端起第3位、第4位以及第5位胺基酸可以爲中性胺基酸，上述中性胺基酸可以如上所述。更具體地，作爲從上述N-末端起第3位胺基酸的絲胺酸可以被異亮胺酸、亮胺酸、蛋胺酸、穀胺醯胺、蘇胺酸、纈胺酸、色胺酸或酪胺酸取代，作爲第4位胺基酸的組胺酸可以被***酸或酪胺酸取代、作爲第5位胺基酸的精胺酸可以被穀胺醯胺、脯胺酸、亮胺酸、蘇胺酸或絲胺酸取代。另一方面，從上述由如序列93所示的胺基酸序列組成的多肽的N-末端起第6位胺基酸可以爲鹼性或中性胺基酸，上述鹼性或中性胺基酸可以如上所述。更具體地，作爲從上述N-末端起第6位胺基酸的脯胺酸可以被丙胺酸、穀胺醯胺、精胺酸、絲胺酸或組胺酸取代。另外，從由如序列93所示的胺基酸序列組成的多肽的N-末端起第7位胺基酸可以爲中性、酸性或鹼性胺基酸，上述中性、酸性或鹼性胺基酸可以如上所述。更具體地，作爲從上述N-末端起第7位胺基酸的絲胺酸可以被天冬胺酸、酪胺酸、甘胺酸、天冬醯胺、蘇胺酸、***酸、色胺酸或精胺酸取代。For example, the 3rd, 4th and 5th amino acids from the N-terminus of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 93 may be neutral amino acids, and the neutral amino acids may be as described above. More specifically, serine as the 3rd amino acid from the N-terminus may be substituted with isoleucine, leucine, methionine, glutamine, threonine, valine, tryptophan or tyrosine, histidine as the 4th amino acid may be substituted with phenylalanine or tyrosine, and arginine as the 5th amino acid may be substituted with glutamine, proline, leucine, threonine or serine. On the other hand, the 6th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence as shown in Sequence 93 can be an alkaline or neutral amino acid, and the alkaline or neutral amino acid can be as described above. More specifically, proline as the 6th amino acid from the N-terminus can be replaced by alanine, glutamine, arginine, serine or histidine. In addition, the 7th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence as shown in Sequence 93 can be a neutral, acidic or alkaline amino acid, and the neutral, acidic or alkaline amino acid can be as described above. More specifically, serine as the 7th amino acid from the N-terminus can be replaced by aspartic acid, tyrosine, glycine, asparagine, threonine, phenylalanine, tryptophan or arginine.
在本發明一實施例中，上述由如序列93所示的胺基酸序列組成的多肽中的5個以下胺基酸被取代的輕鏈CDR2可以爲由分別如序列112至序列135所示的胺基酸序列組成的多肽。In one embodiment of the present invention, the light chain CDR2 in which less than 5 amino acids are substituted in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 93 may be a polypeptide consisting of the amino acid sequences shown in SEQ ID NO: 112 to SEQ ID NO: 135, respectively.
在本發明的抗體或其抗原結合片段中，在由如序列94所示的胺基酸序列組成的多肽中的2個以下胺基酸被取代的輕鏈CDR2中，可以從上述多肽的N-末端起選自由第6位至第8位胺基酸組成的組中的2個以下胺基酸被取代。In the antibody or antigen-binding fragment thereof of the present invention, in the light chain CDR2 in which no more than two amino acids in a polypeptide consisting of an amino acid sequence as shown in SEQ ID NO: 94 are substituted, no more than two amino acids selected from the group consisting of amino acids at positions 6 to 8 from the N-terminus of the above-mentioned polypeptide can be substituted.
例如，從上述由如序列94所示的胺基酸序列組成的多肽的N-末端起第6位以及第8位胺基酸可以爲中性胺基酸，上述中性胺基酸可以如上所述。更具體地，作爲從上述N-末端起第6位以及第8位胺基酸的絲胺酸可以分別被色胺酸以及甘胺酸取代。For example, the 6th and 8th amino acids from the N-terminus of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 94 may be neutral amino acids, and the neutral amino acids may be as described above. More specifically, the 6th and 8th amino acids from the N-terminus may be substituted with tryptophan and glycine, respectively.
在本發明一實施例中，上述由如序列94所示的胺基酸序列組成的多肽中的2個以下胺基酸被取代的輕鏈CDR3可以爲由如序列136所示的胺基酸序列組成的多肽。In one embodiment of the present invention, the light chain CDR3 in which two or less amino acids in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO:94 are substituted may be a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:136.
並且，本發明的抗體或其抗原結合片段可根據需求變形而得。具體地，上述抗體或其抗原結合片段可通過綴合（conjugation）、糖化（glycosylation）、標記或它們的組合來變形。具體地，上述抗體或其抗原結合片段可通過辣根過氧化物酶（HRP，horseradish peroxidase）、鹼性磷酸酶（alkaline phosphatase）、半抗原（hapten）、生物素、鏈酶親和素、螢光物質、放射性物質、量子點、聚乙二醇（PEG，polyethylene glycol）、組胺酸標記等變形。不僅如此，上述抗體或其抗原結合片段還可根據需求來與其他藥物綴合。Furthermore, the antibody or antigen-binding fragment thereof of the present invention can be deformed as required. Specifically, the antibody or antigen-binding fragment thereof can be deformed by conjugation, glycosylation, labeling or a combination thereof. Specifically, the antibody or antigen-binding fragment thereof can be deformed by horseradish peroxidase (HRP), alkaline phosphatase, hapten, biotin, streptavidin, fluorescent substance, radioactive substance, quantum dot, polyethylene glycol (PEG), histidine labeling, etc. In addition, the antibody or antigen-binding fragment thereof can also be conjugated with other drugs as required.
上述抗體或其抗原結合片段可根據普通技術領域中周知的單株抗體製備方法來製備，上述製備方法可由普通技術人員進行適當變形。作為一例，上述抗體可通過在使用從由抗原實現免疫的動物獲取的B淋巴細胞製備雜交瘤來實現製備，或可利用噬菌體顯示技術來製備。The above-mentioned antibody or its antigen-binding fragment can be prepared according to the method for preparing monoclonal antibodies well known in the ordinary technical field, and the above-mentioned preparation method can be appropriately modified by ordinary technicians. As an example, the above-mentioned antibody can be prepared by preparing hybridomas using B lymphocytes obtained from animals immunized with antigens, or can be prepared using phage display technology.
並且，本發明提供對上述抗體或其抗原結合片段進行編碼的核酸。Furthermore, the present invention provides a nucleic acid encoding the above-mentioned antibody or an antigen-binding fragment thereof.
本發明的核酸進行編碼的抗體或其抗原結合片段可具備如上所述的特徵。組成本發明的抗體或其抗原結合片段的胺基酸序列已公知，因而對其進行編碼的核酸序列也對於普通技術人員而言是周知的。並且，只要上述核酸序列維持由此轉譯的抗體或該抗體的抗原片段的活性，則可以是添加、缺失或置換一個或一個以上的鹼基的。The antibody or antigen-binding fragment thereof encoded by the nucleic acid of the present invention may have the above-mentioned characteristics. The amino acid sequence constituting the antibody or antigen-binding fragment thereof of the present invention is known, and thus the nucleic acid sequence encoding it is also known to ordinary technicians. Furthermore, as long as the above-mentioned nucleic acid sequence maintains the activity of the antibody or antigen-binding fragment thereof translated therefrom, one or more bases may be added, deleted or replaced.
並且，本發明提供包含上述核酸的表現載體。Furthermore, the present invention provides an expression vector comprising the above nucleic acid.
本發明的包含在表現載體的核酸可以是對具備如上所述的特徵的抗體或其抗原結合片段進行編碼的。The nucleic acid contained in the expression vector of the present invention may encode an antibody or an antigen-binding fragment thereof having the characteristics as described above.
在本說明書中使用的術語“表現載體（expression vector）”作為在宿主細胞中表現標靶基因的手段，可包括質粒載體、黏粒載體、噬菌體載體、病毒載體等全部。上述表現載體可為了從其所包含的核酸生成肽而包含必要的要素。具體地，上述表現載體可包含信號序列、複製源、標記基因、啟動子、轉錄終止序列等。在此情況下，本發明的抗體或其抗原結合片段被編碼的核酸可與啟動子動作性地相連接。The term "expression vector" used in this specification refers to a means for expressing a target gene in a host cell, and may include all plasmid vectors, cosmid vectors, phage vectors, viral vectors, etc. The above-mentioned expression vector may contain necessary elements for producing a peptide from the nucleic acid contained therein. Specifically, the above-mentioned expression vector may contain a signal sequence, a replication origin, a marker gene, a promoter, a transcription termination sequence, etc. In this case, the nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention may be operably linked to the promoter.
作為一例，用於原核細胞的表現載體可包含進行轉錄的啟動子、用於開始解讀的核糖體結合位置以及轉錄和解讀的終止序列。另一方面，用於真核細胞的表現載體可包含源自哺乳動物或哺乳動物病毒的啟動子以及多腺苷酸化序列。As an example, an expression vector for prokaryotic cells may contain a promoter for transcription, a ribosome binding site for initiation of reading, and a termination sequence for transcription and reading. On the other hand, an expression vector for eukaryotic cells may contain a promoter derived from mammals or mammalian viruses and a polyadenylation sequence.
並且，上述表現載體所包含的標記基因可使用普通技術領域中周知的所有標記基因，具體地，可以是抗生素耐藥性基因。具體地，上述抗生素耐藥性基因可以是包括胺苄西林、艮他黴素、羧比西林、氯黴素、鏈黴素、卡那黴素、新黴素、四環素等在內的對抗生素表現出耐藥性的基因。Furthermore, the marker gene contained in the expression vector may be any marker gene known in the art, and specifically, may be an antibiotic resistance gene. Specifically, the antibiotic resistance gene may be a gene showing resistance to antibiotics including ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, neomycin, tetracycline, etc.
並且，本發明提供包含上述核酸或表現載體的宿主細胞。Furthermore, the present invention provides a host cell comprising the above-mentioned nucleic acid or expression vector.
本發明的包含在宿主細胞的核酸或表現載體可具備如上所述的特徵。作為一例，上述核酸可對本發明的與酸性鞘磷脂酶蛋白特異性結合的抗體或其抗原結合片段進行編碼，上述表現載體可包含如上所述的核酸。The nucleic acid or expression vector contained in the host cell of the present invention may have the above-mentioned characteristics. As an example, the above-mentioned nucleic acid may encode the antibody or antigen-binding fragment thereof that specifically binds to the acid sphingomyelinase protein of the present invention, and the above-mentioned expression vector may contain the above-mentioned nucleic acid.
上述宿主細胞可使用在普通技術領域中可用於抗體或其抗原結合片段生產的所有種類的細胞。具體地，上述宿主細胞可以是原核細胞、酵母或真核細胞。上述原核細胞可包括大腸菌（E.coli）、芽孢桿菌屬菌株、鏈黴菌屬菌株、假單胞菌屬菌株、葡萄球菌屬菌株等，上述酵母可包括釀酒酵母等。另一方面，上述真核細胞可包括COS-7、BHK、CHO、CHOK1、DXB-11、DG-44、CHO/-DHFR、CV1、HEK293、TM4、VERO、HELA、MDCK、BRL 3A、W138、Hep G2、SK-Hep、MMT、TRI、MRC5、FS4、3T3、RIN、A549、PC12、K562、PERC6、SP2/0、NS-0、U20S以及HT1080等。The host cells mentioned above can use all kinds of cells that can be used for producing antibodies or antigen-binding fragments thereof in the ordinary technical field. Specifically, the host cells mentioned above can be prokaryotic cells, yeasts or eukaryotic cells. The prokaryotic cells mentioned above can include Escherichia coli (E. coli), Bacillus strains, Streptomyces strains, Pseudomonas strains, Staphylococcus strains, etc., and the yeasts mentioned above can include brewing yeast, etc. On the other hand, the above-mentioned eukaryotic cells may include COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/-DHFR, CV1, HEK293, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PERC6, SP2/0, NS-0, U20S and HT1080, etc.
並且，上述宿主細胞可以是根據普通技術領域中周知的方法轉染如上所述的核酸或表現載體的。具體地，上述轉染可通過暫態轉染（transient transfection）、微注射、轉導（transduction）、細胞融合、磷酸鈣沉澱法、脂質體介導轉染（liposome-mediated transfection）、DEAE葡聚糖-介導轉染（DEAE dextran-mediated transfection）、凝聚胺-介導轉染（polybrene-mediated transfection）、電穿孔法、基因搶（gene gun）等的方法來執行。並且，上述方法可由普通技術人員進行適當變形。Furthermore, the host cells can be transfected with the nucleic acid or expression vector as described above according to methods well known in the art. Specifically, the transfection can be performed by transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, etc. Furthermore, the above methods can be appropriately modified by ordinary technicians.
進而，本發明提供與酸性鞘磷脂酶蛋白特異性結合的抗體或其抗原結合片段的生產方法，其包括通過培養上述宿主細胞來生產抗體或其抗原結合片段的步驟。Furthermore, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to an acid sphingomyelinase protein, which comprises the step of producing the antibody or the antigen-binding fragment thereof by culturing the above-mentioned host cell.
通過本發明的生產方法生產的抗體或其抗原結合片段可具備如上所述的特徵。The antibody or antigen-binding fragment thereof produced by the production method of the present invention may have the characteristics as described above.
上述培養可根據生產中使用的宿主細胞的種類來使用合適的培養基執行，可根據需求包含適當的補充物。並且，上述培養可根據宿主細胞的種類來在合適的環境下執行。The above-mentioned culture can be performed using a suitable culture medium according to the type of host cell used in production, and can contain appropriate supplements as required. In addition, the above-mentioned culture can be performed in a suitable environment according to the type of host cell.
本發明的生產方法還可包括對由宿主細胞生產的抗體或其抗原結合片段進行回收的步驟。上述回收可根據普通技術領域中周知的方法來執行，這可根據需求來由普通技術人員進行適當變形。作為一例，上述回收可通過利用離心分離或超濾去除雜物並通過層析法等進一步純化所得到的產物來執行。上述層析法可包括親和層析法、負離子層析法、疏水性相互作用層析法等。The production method of the present invention may also include a step of recovering the antibody or antigen-binding fragment thereof produced by the host cell. The above recovery can be performed according to methods known in the art, which can be appropriately modified by ordinary technicians as needed. As an example, the above recovery can be performed by removing impurities by centrifugation or ultrafiltration and further purifying the obtained product by chromatography. The above chromatography may include affinity chromatography, negative ion chromatography, hydrophobic interaction chromatography, etc.
並且，本發明提供包含上述抗體或其抗原結合片段的酸性鞘磷脂酶蛋白檢測用組合物及試劑盒。Furthermore, the present invention provides a composition and a reagent kit for detecting acid sphingomyelinase protein comprising the above-mentioned antibody or an antigen-binding fragment thereof.
本發明的酸性鞘磷脂酶蛋白檢測用組合物及試劑盒中包含的抗體或其抗原結合片段可具備如上所述的特徵。The antibody or antigen-binding fragment thereof contained in the acid sphingomyelinase protein detection composition and reagent kit of the present invention may have the above-mentioned characteristics.
並且，上述組合物可包含與本發明的抗體或其抗原結合片段特異性結合的配體。上述配體可以是發色酵素、螢光物質、放射性同位素或膠體等的檢測體被標示的綴合物以及鏈酶親和素或抗生物素蛋白被處理的配體。除如上所述的試劑之外，本發明的檢測用組合物還可包含能穩定維持它們的結構的蒸餾水或緩衝液。Furthermore, the above composition may contain a ligand that specifically binds to the antibody or antigen-binding fragment thereof of the present invention. The above ligand may be a conjugate labeled with a detector such as a chromogenic enzyme, a fluorescent substance, a radioisotope or a colloid, and a ligand treated with avidin or avidin. In addition to the above reagents, the detection composition of the present invention may also contain distilled water or a buffer solution that can stably maintain their structure.
並且，上述試劑盒可與固體基質相結合，以使得其所包含的抗體或其抗原結合片段的清洗或複合物的分離等的之後步驟變得容易。在此情況下，固體基質可使用合成樹脂、硝化纖維素、玻璃基板、金屬基板、玻璃纖維、微細球體或微細珠。並且，合成樹脂可使用聚酯、聚氯乙烯、聚苯乙烯、聚丙烯、PVDF或尼龍等。Furthermore, the above-mentioned reagent kit may be combined with a solid matrix to facilitate subsequent steps such as washing of the antibody or antigen-binding fragment thereof or separation of the complex. In this case, the solid matrix may be a synthetic resin, nitrocellulose, a glass substrate, a metal substrate, glass fiber, microspheres or microbeads. Furthermore, the synthetic resin may be polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF or nylon.
並且，上述試劑盒可通過普通技術人員周知的現有製造方法進行製造，還可包含緩衝液、穩定化劑、非活性蛋白等。Furthermore, the above-mentioned reagent kit can be manufactured by existing manufacturing methods well known to ordinary technicians, and may also contain a buffer, a stabilizer, an inactive protein, etc.
進而，本發明提供酸性鞘磷脂酶蛋白檢測方法，其包括使上述抗體或其抗原結合片段與試樣產生反應的步驟。Furthermore, the present invention provides a method for detecting acid sphingomyelinase protein, which comprises the step of reacting the above-mentioned antibody or antigen-binding fragment thereof with a sample.
在本發明的酸性鞘磷脂酶蛋白檢測方法中使用的抗體或其抗原結合片段可具備如上所述的特徵。The antibody or antigen-binding fragment thereof used in the method for detecting acid sphingomyelinase protein of the present invention may have the characteristics as described above.
只要是用於檢測酸性鞘磷脂酶蛋白的試樣，則上述試樣可包含所有種類的試樣。並且，利用抗體或其抗原結合片段檢測靶蛋白的方法在普通技術領域中周知，這可由普通技術人員根據需求適當變形執行。The above-mentioned sample may include any kind of sample as long as it is a sample for detecting acid sphingomyelinase protein. In addition, the method of detecting the target protein using an antibody or an antigen-binding fragment thereof is well known in the ordinary technical field, and this can be appropriately modified and performed by ordinary technicians according to needs.
以下，通過以下的實施例詳細說明本發明。但是，以下的實施例僅屬於本發明的例示，本發明並不限定於此。只要形成實際與本發明的發明要求保護範圍中的技術思想相同的結構並提供相同的作用效果，則將屬於本發明的技術範圍。The present invention is described in detail below through the following embodiments. However, the following embodiments are merely illustrative of the present invention, and the present invention is not limited thereto. As long as a structure that is actually the same as the technical idea in the scope of protection of the invention claimed by the present invention is formed and the same effect is provided, it will belong to the technical scope of the present invention.
實施例Embodiment 11 ．製備與酸性神經鞘磷脂酶（． Preparation and acidic neurosphingomyelinase ( acid sphingomyelinaseacid sphingomyelinase ，, ASMASM ）蛋白特異性結合的抗體) Protein-specific binding antibodies
#9104抗體爲與人酸性神經鞘磷脂酶蛋白（序列139）特異性結合的抗體，從#9104抗體製備其變體。Antibody #9104 is an antibody that specifically binds to human acidic sphingomyelinase protein (sequence 139), and its variants are prepared from antibody #9104.
首先，利用隨機引物通過常規方法擴增#9104抗體的VH以及VL基因，以誘導#9104抗體的重鏈以及輕鏈CDR序列中的隨機突變。將每個擴增的VH以及VL基因連接至小鼠的重鏈恒定區1（CH1，序列142）以及輕鏈恒定區（CL，序列143）並以scFab形式***pComb3xss噬菌粒載體中。利用其製備#9104隨機突變文庫（random mutagenesis library），並利用上述文庫通過常規方法進行篩選以選擇與人酸性神經鞘磷脂酶蛋白特異性結合的scFab。所選的scFab在其重鏈可變區以及輕鏈可變區各自的羧基末端（carboxy terminus）製備表現載體，使得由分別如序列137或序列138所示的鹼基序列組成的人重鏈恒定區（constant region）以及人輕鏈λ恒定區以連接的形式表現。結果，將所選的scFab的重鏈可變區的胺基酸序列以及核酸序列示於下述表1中，將輕鏈可變區的胺基酸序列以及核酸序列示於下述表2中。First, random primers were used to amplify the VH and VL genes of the #9104 antibody by conventional methods to induce random mutations in the heavy chain and light chain CDR sequences of the #9104 antibody. Each amplified VH and VL gene was linked to the mouse heavy chain constant region 1 (CH1, sequence 142) and light chain constant region (CL, sequence 143) and inserted into the pComb3xss phagemid vector in the form of scFab. The #9104 random mutagenesis library was prepared using it, and the above library was screened by conventional methods to select scFabs that specifically bind to human acidic sphingomyelinase protein. The selected scFab was prepared to express a vector at the carboxy terminus of each of the heavy chain variable region and the light chain variable region so that a human heavy chain constant region (constant region) consisting of the base sequence shown in SEQ ID NO: 137 or SEQ ID NO: 138 and a human light chain λ constant region were expressed in a linked form. As a result, the amino acid sequence and nucleic acid sequence of the heavy chain variable region of the selected scFab are shown in Table 1 below, and the amino acid sequence and nucleic acid sequence of the light chain variable region are shown in Table 2 below.
表1
表2
在此情况下，表現載體使用哺乳類表現載體（mammalian expression vector）。將所製備的表現載體轉染至ExpiCHO細胞系以製備與人酸性神經鞘磷脂酶蛋白結合的全長抗體。In this case, a mammalian expression vector is used as the expression vector. The prepared expression vector is transfected into ExpiCHO cell line to prepare a full-length antibody that binds to human acidic sphingomyelinase protein.
實施例Embodiment 22 ．决定互補决定區（． Determine the complementary decision area ( complementarity determining regioncomplementarity determining region ，, CDRCDR ））
通過常規方法確認所製備的上述scFv中的互補决定區，結果，將重鏈可變區的CDR序列示於表3中，將輕鏈可變區的CDR序列示於表4中。The complementation determining regions in the prepared scFv were confirmed by conventional methods. As a result, the CDR sequences of the heavy chain variable region are shown in Table 3, and the CDR sequences of the light chain variable region are shown in Table 4.
表3
表4
如表3以及表4所示，確認到重鏈可變區的CDR1、CDR3以及輕鏈可變區的CDR1與#9104抗體具有相同的序列，相反，重鏈可變區的CDR2、輕鏈可變區的CDR2以及CDR3具有構成其的部分胺基酸被取代的序列。As shown in Tables 3 and 4, it was confirmed that CDR1 and CDR3 of the heavy chain variable region and CDR1 of the light chain variable region had the same sequence as that of the #9104 antibody, whereas CDR2 of the heavy chain variable region, CDR2 and CDR3 of the light chain variable region had sequences in which some of the amino acids constituting them were substituted.
實驗例Experimental example 11 ．確認與酸性神經鞘磷脂酶蛋白的結合親和性． Confirmation of binding affinity to acidic sphingomyelinase protein
利用Octet ®QK384系統（頗爾生命科學（Pall Life Sciences））測定上述中製備的與酸性神經鞘磷脂酶蛋白特異性結合的抗體與酸性神經鞘磷脂酶蛋白的結合親和性以及相互作用動力學。 The Octet ® QK384 system (Pall Life Sciences) was used to measure the binding affinity and interaction kinetics of the antibody prepared above that specifically binds to acidic sphingomyelinase protein and acidic sphingomyelinase protein.
首先，利用抗人IgG Fc捕獲（AHC）生物傳感器捕獲上述實施例1中製備的與酸性神經鞘磷脂酶蛋白特異性結合的抗體，然後添加1.25nM、2.5nM、5nM、10nM或20nM的重組人酸性神經鞘磷脂酶蛋白溶液。添加人酸性神經鞘磷脂酶蛋白溶液，並觀察反應物的結合相約1200秒，然後添加1×動力學緩衝液（kinetics buffer）（ForteBio公司），並觀察反應物的分離相約1500秒。利用Octet ®分析軟體（頗爾生命科學（Pall Life Sciences））測定各抗體的吸附率常數（association constant：K a）、分離率常數（dissociation constant：K d）以及平衡解離常數（equilibrium dissociation constant：K D）。結果，將#9104抗體中如表1所示的重鏈CDR部分突變的抗體對人酸性神經鞘磷脂酶蛋白的結合力示於表5中，將如表2所示的輕鏈CDR部分突變的抗體對人酸性神經鞘磷脂酶蛋白的結合力示於表6中。 First, the antibody specifically binding to the acidic sphingomyelinase protein prepared in Example 1 was captured using an anti-human IgG Fc capture (AHC) biosensor, and then a 1.25nM, 2.5nM, 5nM, 10nM or 20nM recombinant human acidic sphingomyelinase protein solution was added. The human acidic sphingomyelinase protein solution was added, and the binding of the reactants was observed for about 1200 seconds, and then 1× kinetics buffer (ForteBio) was added, and the separation of the reactants was observed for about 1500 seconds. The association constant ( Ka ), dissociation constant (Kd), and equilibrium dissociation constant ( KD ) of each antibody were measured using Octet® analysis software (Pall Life Sciences). The results show that the binding ability of the #9104 antibody with the heavy chain CDR partial mutation shown in Table 1 to human acidic sphingomyelinase protein is shown in Table 5, and the binding ability of the antibody with the light chain CDR partial mutation shown in Table 2 to human acidic sphingomyelinase protein is shown in Table 6.
表5
表6
如表5以及表6所示，實施例1中製備的抗體與人酸性神經鞘磷脂酶蛋白以10 -10M至10 -9M水平的結合力結合。 As shown in Table 5 and Table 6, the antibody prepared in Example 1 binds to human acidic sphingomyelinase protein at a binding affinity of 10 -10 M to 10 -9 M.
實驗例Experimental example 22 ．組合重鏈以及輕鏈可變區． Combine heavy chain and light chain variable area
通過組合上述中所選的#9104抗體的8種重鏈可變區以及5種輕鏈可變區來製備40種抗體（表7）。40 antibodies were prepared by combining the 8 heavy chain variable regions and 5 light chain variable regions of the #9104 antibody selected above (Table 7).
表7
如上所述，在ExpiCHO細胞系中小規模表現後，如實施例1所述地確認對人酸性神經鞘磷脂酶蛋白的結合力。在此情况下，由#9104v-H33重鏈可變區以及#9104v-L23輕鏈可變區的組合製備的抗體未表現，因此從與酸性神經鞘磷脂酶蛋白結合力分析中排除。結果，將上述抗體對人酸性神經鞘磷脂酶蛋白的結合力示於表8中。As described above, after small-scale expression in the ExpiCHO cell line, the binding ability to human acidic sphingomyelinase protein was confirmed as described in Example 1. In this case, the antibody prepared by the combination of #9104v-H33 heavy chain variable region and #9104v-L23 light chain variable region was not expressed, and was therefore excluded from the analysis of binding ability to acidic sphingomyelinase protein. As a result, the binding ability of the above antibodies to human acidic sphingomyelinase protein is shown in Table 8.
表8
如表8所示，所製備的抗體與人酸性神經鞘磷脂酶蛋白以10 -10M至10 -9M水平的結合力結合。 As shown in Table 8, the prepared antibodies bound to human acidic sphingomyelinase protein at a binding affinity of 10 -10 M to 10 -9 M.
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